Culture vessels containing 3d cell culture substrates with diffusion structures

Abstract

A cell culture device comprises a multi-well cell culture plate comprising a plurality of wells, each well comprising a top, a bottom, and a sidewall disposed between the top and the bottom and having an interior surface comprising an ultra-low attachment surface. A plurality of scaffolds are disposed within wells of the multi-well cell culture plate, each scaffold comprising a cell-adherent surface. In some embodiments, the scaffold comprises a fiber scaffold. In some embodiments, the scaffold comprises an artificial vascular scaffold. In some embodiments, cell culture devices comprise a plurality of hydrogel scaffolds disposed in a multi-well cell culture plate, the plurality of hydrogel scaffolds comprising hydrogel fibers of differing lengths, wherein opposite ends of a hydrogel fiber are disposed in different wells within the multi-well cell culture plate to create interconnected wells.

What is claimed is: 
     
         1 . A cell culture device comprising:
 a multi-well cell culture plate comprising a plurality of wells, each well comprising a top aperture, a bottom, and a sidewall disposed between the top and the bottom, wherein each well is configured with an interior surface comprising a cell non-adherent surface; and   a plurality of scaffolds disposed within the plurality of wells of the multi-well cell culture plate, wherein at least one scaffold is disposed in each well and each scaffold comprising a cell-adherent surface.   
     
     
         2 . The device of  claim 1 , wherein the bottom comprises a hemispherical shape. 
     
     
         3 . (canceled) 
     
     
         4 . The device of  claim 1 , wherein at least one end of the scaffold is anchored to a bottom portion of each well. 
     
     
         5 . The device of  claim 1 , wherein the bottom comprises a plurality of microcavities. 
     
     
         6 . The device of  claim 5 , wherein a scaffold is anchored to a bottom portion of each microcavity of the plurality of microcavities. 
     
     
         7 . (canceled) 
     
     
         8 . The device of  claim 6 , wherein the scaffold has an average length in a range of 100 μm to 1000 μm. 
     
     
         9 . The device of  claim 6 , wherein the scaffold has an average width in a range of 10 μm to 100 μm. 
     
     
         10 . The device of  claim 6 , wherein a scaffold of the plurality of scaffolds comprises a fiber scaffold. 
     
     
         11 . The device of  claim 10 , wherein the fiber scaffold is formed from polyvinylalcohol, polyacrylamide, polyvinylpyrrolidone, poly(2-hydroxyethyl methacrylate), polystyrene, polypropylene, polygalacturonic acid, and/or combinations thereof. 
     
     
         12 . The device of  claim 11 , wherein the fiber scaffold comprises a plurality of fibers. 
     
     
         13 . The device of  claim 12 , wherein each fiber in the plurality of fibers is anchored to a bottom portion of each well. 
     
     
         14 . The device of  claim 13 , wherein anchored ends of individual fibers are distanced 100 μm to 200 μm apart from one another in each well. 
     
     
         15 . The device of  claim 1 , wherein a scaffold of the plurality of scaffolds comprises an artificial vascular scaffold. 
     
     
         16 . The device of  claim 15 , wherein the artificial vascular scaffold comprises a hollow fiber. 
     
     
         17 . The device of  claim 15 , wherein the artificial vascular scaffold comprises a plurality of hollow fibers. 
     
     
         18 . The device of  claim 17 , wherein individual hollow fibers are anchored to the bottom of a well 100 μm to 200 μm from one another. 
     
     
         19 . The device of  claim 16 , wherein the hollow fiber is formed from a non-ionic polymer. 
     
     
         20 . The device of  claim 19 , wherein the non-ionic polymer comprises polyvinylalcohol, polyacrylamide, polyvinylpyrrolidone, poly(2-hydroxyethyl methacrylate), polystyrene, polypropylene, or polygalacturonic acid. 
     
     
         21 . The device of  claim 16 , wherein the hollow fiber further comprises a hydrogel coating. 
     
     
         22 . The device of  claim 15 , wherein the artificial vascular scaffold comprises a hydrogel. 
     
     
         23 . A method of culturing three-dimensional (3D) cell cultures, the method comprising:
 seeding cells in a cell culture device according to  claim 1 , wherein the cells attach to the cell adherent surface of each scaffold disposed within the cell culture device; and   culturing the cells into a 3D cell culture by adding cell culture medium to the cell culture device to provide nutrients and oxygen, wherein the cells remain attached to each scaffold during addition or exchange of cell culture medium; and   optionally digesting each of the plurality of scaffolds.   
     
     
         24 . The method of  claim 23 , further comprising imaging 3D cell cultures attached to each of the plurality of scaffolds of the cell culture device. 
     
     
         25 . An interconnected cell culture device comprising:
 a multi-well cell culture plate comprising a plurality of wells, each well comprising a top, a bottom, and a sidewall disposed between the top and the bottom, wherein each well is configured with an interior surface comprising a cell non-adherent surface; and   a plurality of hydrogel scaffolds disposed in the multi-well cell culture plate, the plurality of hydrogel scaffolds comprising hydrogel fibers of differing lengths, wherein opposite ends of a hydrogel fiber are disposed in different wells within the multi-well cell culture plate to create interconnected wells.   
     
     
         26 . The device of  claim 25 , wherein the bottom comprises a hemispherical shape. 
     
     
         27 . The device of  claim 25 , wherein the hydrogel fibers have lengths in a range of 100 μm to 100 mm. 
     
     
         28 . The device of  claim 27 , wherein the hydrogel fibers have average widths of 10 μm. 
     
     
         29 . The device of  claim 28 , wherein each hydrogel fiber comprises a cell-adherent surface. 
     
     
         30 . The device of  claim 25 , wherein the hydrogel fibers are formed of extracelluar matrix (ECM) proteins, decellularized tissue ECM scaffolds, ECM peptide binding sequences, crosslinked polymers, and/or combinations thereof. 
     
     
         31 . The device of  claim 25 , wherein the hydrogel scaffolds are unanchored or free-floating within the wells. 
     
     
         32 . The device of  claim 25 , wherein the bottom comprises a plurality of microcavities. 
     
     
         33 . The device of  claim 32 , wherein opposite ends of hydrogel fibers are disposed in different microcavities to create interconnected microcavities. 
     
     
         34 . A method of forming an interconnected cell culture device, the method comprising:
 seeding a cell culture device with cells, the cell culture device comprising a multi-well cell culture plate comprising a plurality of wells, each well comprising a top, a bottom, and a sidewall disposed between the top and the bottom and having an interior surface comprising an ultra-low attachment surface;   seeding the cell culture device with a plurality of hydrogel scaffolds, the hydrogel scaffolds comprising a plurality of hydrogel fibers, wherein opposite ends of a hydrogel fiber are disposed in different wells within the multi-well cell culture plate to create interconnected wells; and   providing cell culture medium to provide nutrients and oxygen for cell growth and formation of three-dimensional (3D) cell cultures.   
     
     
         35 . The method of  claim 34 , wherein the bottom comprises a hemispherical shape. 
     
     
         36 . The method of  claim 34 , wherein the bottom comprises a plurality of microcavities.